close

Вход

Забыли?

вход по аккаунту

?

Inactivation of the cannabinoid receptor CB1 prevents leukocyte infiltration and experimental fibrosis.

код для вставкиСкачать
ARTHRITIS & RHEUMATISM
Vol. 62, No. 11, November 2010, pp 3467–3476
DOI 10.1002/art.27642
© 2010, American College of Rheumatology
Inactivation of the Cannabinoid Receptor CB1 Prevents
Leukocyte Infiltration and Experimental Fibrosis
Sieglinde Marquart,1 Pawel Zerr,1 Alfiya Akhmetshina,1 Katrin Palumbo,1 Nicole Reich,1
Michal Tomcik,2 Angelika Horn,1 Clara Dees,1 Matthias Engel,1 Jochen Zwerina,1
Oliver Distler,3 Georg Schett,1 and Jörg H. W. Distler1
Objective. Cannabinoids are derivates of the marijuana component ⌬9-tetrahydrocannabinol that exert
their effects on mesenchymal cells and immune cells via
CB1 and CB2 receptors. The aim of the present study
was to evaluate the role of CB1 in systemic sclerosis.
Methods. CB1-deficient (CB1ⴚ/ⴚ) mice and wildtype littermates (CB1ⴙ/ⴙ mice) were injected with bleomycin. CB1 signaling was activated in vivo with the
selective agonist N-(2-chloroethyl)-5Z,8Z,11Z,14Zeicosatetraenamide (ACEA). Bone marrow transplantation experiments were performed to investigate whether
the phenotype of CB1ⴚ/ⴚ mice was mediated by leukocytes or mesenchymal cells. The role of CB1 was also
investigated in the TSK-1 mouse model.
Results. CB1 ⴚ/ⴚ mice were protected from
bleomycin-induced dermal fibrosis, with reduced dermal thickening, hydroxyproline content, and myofibroblast counts. Inactivation of CB1 decreased the number
of infiltrating T cells and macrophages in lesional skin.
In contrast, activation of CB1 with ACEA increased
leukocyte infiltration and enhanced the fibrotic response to bleomycin. The phenotype of CB1ⴚ/ⴚ mice was
mimicked by transplantation of CB1ⴚ/ⴚ mouse bone
marrow into CB1ⴙ/ⴙ mice, demonstrating that CB1
exerts its profibrotic effects indirectly by regulating
leukocyte infiltration. Consistently, knockdown of CB1
did not prevent fibrosis in the inflammationindependent TSK-1 mouse model.
Conclusion. We demonstrate that the cannabinoid
receptor CB1 is crucial for leukocyte infiltration and
secondary fibroblast activation and that inactivation of
CB1 exerts potent antifibrotic effects in inflammationdriven models of fibrosis.
Systemic sclerosis (SSc) is a connective tissue
disease of unknown etiology that affects the skin and a
variety of internal organs such as the lungs, the heart,
and the gastrointestinal tract. Early stages of SSc are
characterized by vascular changes and inflammatory
infiltrates in involved organs (1). The inflammatory infiltrates are dominated by macrophages and activated
T cells. Later stages of SSc are characterized by an
excessive accumulation of extracellular matrix components. The resulting fibrosis disrupts the physiologic
tissue structure and frequently leads to dysfunction of
the affected organs. The accumulation of extracellular
matrix results from an increased release of collagen and
other components of the extracellular matrix by pathologically activated SSc fibroblasts (2). Activated fibroblasts that have differentiated into myofibroblasts and
release excessive amounts of collagen are mostly localized adjacent to inflammatory infiltrates, suggesting that
leukocytes may trigger the initiation of fibrosis in early
stages of SSc (3). However, the molecular pathways that
regulate leukocyte infiltration and subsequent fibroblast
activation are incompletely understood (1).
Supported by the Interdisciplinary Center of Clinical Research (IZKF) in Erlangen (grant A20) and by the Deutsche Forschungsgesellschaft. Dr. J. H. W. Distler is recipient of the Career
Support Award of Medicine of the Ernst Jung Foundation.
1
Sieglinde Marquart, Pawel Zerr, MSc, Alfiya Akhmetshina,
PhD, Katrin Palumbo, MSc, Nicole Reich, Angelika Horn, Clara Dees,
Matthias Engel, MD, Jochen Zwerina, MD, Georg Schett, MD, Jörg
H. W. Distler, MD: University of Erlangen–Nuremberg, Erlangen,
Germany; 2Michal Tomcik, MD: University of Erlangen–Nuremberg,
Erlangen, Germany, and Charles University, Prague, Czech Republic;
3
Oliver Distler, MD: University Hospital Zurich, Zurich, Switzerland.
Dr. O. Distler has received consulting fees, speaking fees,
and/or honoraria from Actelion, Pfizer, Encysive, Fibrogene, ErgoNex,
NicOx, Biozentrum, and Bristol-Myers Squibb (less than $10,000 each).
Dr. J. H. W. Distler has received consulting fees, speaking fees, and/or
honoraria from Actelion, Pfizer, Encysive, Ergonex, NiCox, and BristolMyers Squibb (less than $10,000 each).
Address correspondence and reprint requests to Jörg H. W.
Distler, MD, Department of Internal Medicine 3 and Institute for Clinical
Immunology, Universitätsstrasse 29, University of Erlangen–Nuremberg,
91054 Erlangen, Germany. E-mail: joerg.distler@uk-erlangen.de.
Submitted for publication March 11, 2010; accepted in revised
form June 29, 2010.
3467
3468
Cannabinoids are derivates of the marijuana
component ⌬9-tetrahydrocannabinol. They can be classified into 3 different groups according to their origin.
The family of cannabinoids includes endogenous cannabinoids (endocannabinoids) that are synthesized within
the human body, plant-derived cannabinoids like the
lead compound ⌬9-tetrahydrocannabinol, and synthetic
cannabinoids synthesized for therapeutic interventions
(4). Unlike early hypotheses, the effects of cannabinoids
are not restricted to neurons and to the central nervous
system, but cannabinoids affect many cell types (4,5).
Cannabinoids are currently being evaluated for the
treatment of different tumors, since they inhibit tumor
cell proliferation and induce cell cycle arrest in transformed cells (6). Cannabinoids are also crucial for bone
homeostasis, since they control the differentiation and
proliferation of osteoclast precursor cells and osteoblasts
(7). Moreover, cannabinoids have been implicated in the
regulation of immune responses by controlling leukocyte
activation, cytokine release, and chemotaxis (8).
These pleiotropic effects are mediated by 2 different cannabinoid receptors, CB1 and CB2 (9,10). Despite similarities in the encoding nucleotide sequence,
the expression patterns of CB1 and CB2 are distinct and
they mediate different or even opposing effects. Striking
examples of the distinct roles of CB1 and CB2 are their
effects on bone mass. Inactivation of CB1 increases the
bone mass, whereas inactivation of CB2 accelerates agerelated trabecular bone loss (11,12). The different effects of CB1 and CB2 have stimulated the development
of highly selective agonists and antagonists for both
receptors, and synthetic and selective CB1 antagonists
are available that have been proven to potently inhibit
CB1 signaling in humans (13,14).
We demonstrated recently that CB2 exerts antifibrotic effects in the mouse model of bleomycininduced fibrosis (15). Inactivation of CB2 resulted in
increased accumulation of collagen and more pronounced
dermal thickening, whereas activation of CB2 effectively
prevented bleomycin-induced fibrosis. Intrigued by the
opposing roles of CB1 and CB2 in bone homeostasis, we
aimed to investigate the role of CB1 in fibroblast activation and fibrosis. We demonstrate that, in contrast to CB2,
activation of CB1 results in exacerbation of fibrosis,
whereas inactivation of CB1 exerts potent antifibrotic
effects. Inactivation of CB1 reduced leukocyte infiltration
into lesional skin and prevented subsequent fibroblast
activation and collagen accumulation upon challenge with
bleomycin.
MARQUART ET AL
MATERIALS AND METHODS
Bleomycin-induced dermal fibrosis in CB1-deficient
mice. Mice deficient for CB1 (CB1⫺/⫺ mice) (16) were backcrossed onto a C57BL/6 background for at least 6 generations.
Wild-type (WT) C57BL/6 littermates expressing CB1 (CB1⫹/⫹
mice) were used as controls. Skin fibrosis was induced in
6-week-old male mice by local injections of bleomycin for 4
weeks as described (17). Briefly, 100 ␮l of bleomycin dissolved
in 0.9% NaCl at a concentration of 0.5 mg/ml was administered
every other day by subcutaneous injections in defined areas of
1 cm2 at the upper back. Subcutaneous injections of 100 ␮l
0.9% NaCl were used as control treatment. Four different
groups were analyzed, consisting of 2 groups with CB1⫺/⫺ mice
and 2 groups with CB1⫹/⫹ mice. One group of CB1⫺/⫺ mice
and 1 group of CB1⫹/⫹ mice were challenged with bleomycin,
while the remaining 2 groups were injected with NaCl. After
4 weeks, mice were killed by cervical dislocation. The 4 groups
consisted of 20 mice in total. All animal experiments were
approved by the local ethics committee.
Activation of CB1 in experimental fibrosis. To maximally activate CB1 signaling in experimental fibrosis, C57BL/6
mice challenged with bleomycin were additionally treated with
N-(2-chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (ACEA).
ACEA is a highly selective CB1 receptor agonist with a Ki value
of 1.4 nM and selectivity for CB1 receptors ⬎1,400-fold that for
CB2 receptors (18,19). ACEA was purchased from Biozol and
dissolved in anhydrous ethanol at a concentration of 10 mg/ml.
The working solutions were prepared fresh on the day of the
experiments by diluting the stock solutions in NaCl. ACEA was
administered by intraperitoneal injections twice a day at a concentration of 7.5 mg/kg for 4 weeks. Treatment with ACEA
started in parallel to bleomycin challenge. Mice injected with
NaCl and with bleomycin only were used as controls. Fifteen mice
were analyzed in these experiments.
Bone marrow transplantation. To analyze the role of
bone marrow–derived cells and fibroblasts in the phenotype of
CB1⫺/⫺ mice, bone marrow transplantation experiments were
performed (15). Female CB1⫺/⫺ and CB1⫹/⫹ mice served as
donors of bone marrow. Tibias and femurs were prepared
under sterile conditions. Bone marrow cells were flushed from
the bone marrow cavities with phosphate buffered saline (PBS)
and subsequently filtered through 70-␮m nylon meshes (BD
Biosciences). Erythrocytes were hemolyzed, and the remaining
bone marrow cells were kept on ice until the time of transplantation. For transplantation, 2.0 ⫻ 106 bone marrow cells of
donor mice were resuspended in 0.1 ml PBS and injected via
the tail veins. Male CB1⫺/⫺ or CB1⫹/⫹ mice received bone
marrow transplants at age 4 weeks. Recipient CB1⫺/⫺ or
CB1⫹/⫹ mice underwent whole body irradiation at a dose of 4
Gray twice before transplantation to irradiate their bone
marrow. Two weeks after bone marrow transplantation, when
stable engraftment had occurred, mice were challenged with
bleomycin for 4 weeks as described above. Twenty-six mice
were analyzed in these experiments.
Inactivation of CB1 in TSK-1 mice. To investigate the
role of CB1 in a noninflammatory model of SSc, CB1⫺/⫺ mice
were crossed with TSK-1 mice to generate TSK-1 mice deficient for CB1 (CB1⫺/⫺ TSK-1 mice). The TSK-1 phenotype is
caused by a dominant mutation in the fibrillin 1 gene (20).
TSK-1 mice are characterized by accumulation of collagen
ANTIFIBROTIC EFFECTS OF CB1 INACTIVATION
3469
Figure 1. CB1-deficient (CB1⫺/⫺) mice are protected from dermal fibrosis. A, Reduced accumulation of collagen and dermal fibrosis
in CB1⫺/⫺ mice upon challenge with bleomycin. Representative sections are shown (hematoxylin and eosin stained; original
magnification ⫻ 100). B, Decreased dermal thickening in CB1⫺/⫺ mice compared with their wild-type CB1⫹/⫹ littermates. C, Reduced
hydroxyproline content in lesional skin of CB1⫺/⫺ mice. D, Lower myofibroblast counts in CB1⫺/⫺ mice. CB1⫹/⫹ control mice were
injected with NaCl, and the values for these mice were set at 1.0; the other results were normalized to this value. Values are the mean ⫾
SEM. ⴱ ⫽ P ⱕ 0.05 versus CB1⫹/⫹ mice challenged with bleomycin.
fibers in the hypodermis, resulting in progressive hypodermal
thickening. In contrast to bleomycin-induced fibrosis, inflammatory infiltrates are absent, and the aberrant activation of
fibroblasts is not caused by the release of inflammatory
mediators from leukocytes. Similar to SSc fibroblasts, fibroblasts from TSK-1 mice are endogenously activated, with an
increased release of collagen that persists for several passages
in vitro. Thus, TSK-1 mice are a model for later, inflammationindependent stages of SSc, whereas bleomycin-induced fibrosis
represents early, inflammation-dependent stages of SSc.
Genotyping of TSK-1 mice was performed using polymerase chain reaction with the following primers: mutated
fibrillin 1/TSK-1, 5⬘-GTTGGCAACTATACCTGCAT-3⬘ (forward) and 5⬘-CCTTTCCTGGTAACATAGGA-3⬘ (reverse).
Four groups with a total of 23 mice were analyzed. One group
consisted of TSK-1 mice expressing CB1 (CB1⫹/⫹). A second
group consisted of TSK-1 mice deficient for CB1 (CB1⫺/⫺).
The last 2 groups consisted of pa/pa (control) mice, one group
expressing CB1 and the other deficient for CB1. Mice were
killed by cervical dislocation at age 10 weeks to analyze the
hypodermal thickness, the hydroxyproline content, and the
number of myofibroblasts in lesional skin.
Histologic analysis. Lesional skin areas were excised,
fixed in 4% formalin, and embedded in paraffin. Five
micrometer–thick sections were stained with hematoxylin and
eosin. The dermal thickness was analyzed at 100⫻ magnification by measuring the distance between the epidermal–dermal
junction and the dermal–subcutaneous fat junction at 4 sites
from the lesional skin of each mouse (21). Infiltrating leukocytes in lesional skin of CB1⫺/⫺ mice, CB1⫹/⫹ mice, CB1⫹/⫹
mice treated with ACEA, and bone marrow–transplanted mice
were quantified on hematoxylin and eosin–stained sections.
Eight different high-power fields from different tissue sites
from each mouse were evaluated for polymorphonuclear cells
at 400⫻ magnification by an experienced examiner (JHWD)
who was blinded to the treatment. Images were captured with
an Eclipse 80i microscope (Nikon) equipped with a DSP
3CCD camera (Sony).
Hydroxyproline assay. The collagen content in lesional
skin samples was explored by hydroxyproline assay (22). After
digestion of punch biopsy samples (3 mm in diameter) in 6M
HCl for 3 hours at 120°C, the pH of the samples was adjusted
to 7 with 6M NaOH. Afterward, samples were mixed with
0.06M chloramine T and incubated for 20 minutes at room
temperature. Next, 3.15M perchloric acid and 20%
p-dimethylaminobenzaldehyde were added, and samples were
incubated for an additional 20 minutes at 60°C. The absorbance was determined at 557 nm with a SpectraMax 190
microplate spectrophotometer (Molecular Devices).
Immunohistochemistry for ␣-smooth muscle actin
(␣-SMA), CD3, and F4/80. The expression of ␣-SMA, T cells,
and macrophages was quantified in paraffin-embedded sections of lesional skin from CB1⫺/⫺ mice, CB1⫹/⫹ mice,
CB1⫹/⫹ mice treated with ACEA, and bone marrow–
transplanted mice. Myofibroblasts were identified by staining
for ␣-SMA as described (23,24). After deparaffinization and
blocking with 5% horse serum and 3% H2O2, skin sections
were incubated with anti–␣-SMA antibodies (clone 1A4; SigmaAldrich). Polyclonal rabbit anti-mouse antibodies labeled with
horseradish peroxidase (HRP) (Dako) were used as secondary
antibodies. To quantify the numbers of infiltrating T cells, skin
sections were stained for CD3. After deparaffinization, antigen
retrieval with Tris–EDTA–Tween, and blocking with 10% goat
serum and 0.3% H2O2, sections were incubated with rabbit
polyclonal anti-CD3 antibodies (Abcam). Polyclonal HRPlabeled goat anti-rabbit Ig (Dako) were used as secondary
3470
MARQUART ET AL
Figure 2. Treatment with the CB1 agonist N-(2-chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (ACEA) increases sensitivity to
bleomycin-induced fibrosis. A, Exacerbation of dermal fibrosis in bleomycin-injected CB1⫹/⫹ mice upon treatment with ACEA
compared with mice injected only with bleomycin. Representative sections are shown (hematoxylin and eosin stained; original
magnification ⫻ 100). B–D, Bleomycin-induced fibrosis in CB1⫹/⫹ mice aggravated by more pronounced dermal thickening (B),
increased hydroxyproline content (C), and higher myofibroblast counts (D) in lesional skin of ACEA-treated mice. CB1⫹/⫹ control
mice were injected with NaCl, and the values for these mice were set at 1.0; the other results were normalized to this value. Values
are the mean ⫾ SEM. ⴱ ⫽ P ⱕ 0.05 versus CB1⫹/⫹ mice challenged with bleomycin alone.
antibodies. Macrophages were detected by staining for F4/80.
After antigen retrieval with proteinase K and blocking, sections were incubated with rat anti-mouse F4/80 antibodies
(AbD Serotec). Alkaline phosphatase–labeled polyclonal goat
anti-rat antibodies (Abcam) served as secondary antibodies.
Irrelevant isotype antibodies were used for controls.
Staining was visualized with 3,3⬘-diaminobenzidine peroxidase
substrate solution (Sigma-Aldrich) (for ␣-SMA and CD3) or
with the use of a BCIP/nitroblue tetrazolium Alkaline Phosphatase Substrate Kit IV (Vector) (for F4/80). Sections stained
for ␣-SMA and F4/80 were counterstained with hematoxylin.
The number of myofibroblasts was determined at 200⫻ magnification in 4 different sections from each mouse. T cells and
macrophages were counted in 8 different sections of lesional
skin of each mouse at 400⫻ magnification. Counting was
performed in a blinded manner by an experienced examiner
(JHWD).
Statistical analysis. Data are expressed as the mean ⫾
SEM. The Mann-Whitney U test was used for statistical
analyses. P values less than or equal to 0.05 were considered
significant.
RESULTS
CB1ⴚ/ⴚ mice are protected from bleomycininduced dermal fibrosis. To evaluate the role of CB1 in
fibrosis, CB1⫺/⫺ mice and their CB1⫹/⫹ littermates were
challenged with bleomycin. No differences in skin archi-
tecture were observed between CB1⫺/⫺ mice and
CB1⫹/⫹ mice injected with NaCl (Figure 1A). However,
CB1⫺/⫺ mice were protected from bleomycin-induced
fibrosis. Dermal thickening upon bleomycin challenge
was reduced by (mean ⫾ SEM) 62 ⫾ 3% in CB1⫺/⫺
mice compared with CB1⫹/⫹ mice (P ⫽ 0.01) (Figure
1B). Consistently, the hydroxyproline content in lesional
skin of CB1⫺/⫺ mice was significantly lower than that in
lesional skin of CB1⫹/⫹ mice, with a decrease of 57 ⫾
6% (P ⫽ 0.05) (Figure 1C). Myofibroblast counts were
also reduced by 62 ⫾ 6% in CB1⫺/⫺ mice upon bleomycin challenge (P ⫽ 0.01) (Figure 1D). Together, these
data demonstrate that inactivation of CB1 protects
against fibrosis.
Activation of CB1 signaling exacerbates experimental fibrosis. The effects of an increased activation of
CB1 on experimental fibrosis were evaluated using the
highly selective CB1 agonist ACEA (19). Treatment
with ACEA during bleomycin challenge resulted in
exacerbation of dermal fibrosis (Figure 2A). Dermal
thickening was 40 ⫾ 5% more pronounced in ACEAtreated mice than in mice injected with bleomycin alone
(P ⫽ 0.01) (Figure 2B). Consistently, the hydroxyproline
content and the number of myofibroblasts were also
ANTIFIBROTIC EFFECTS OF CB1 INACTIVATION
3471
Figure 3. CB1 regulates leukocyte infiltration into lesional skin. Inflammation is reduced in CB1-deficient (CB1⫺/⫺) mice challenged
with bleomycin, as shown by decreased leukocyte counts (A) with significantly lower numbers of infiltrating T cells (B) and
macrophages (C) in lesional skin of CB1⫺/⫺ mice than in that of CB1⫹/⫹ mice. CB1⫹/⫹ control mice were injected with NaCl, and the
values for these mice were set at 1.0; the other results were normalized to this value. Values are the mean ⫾ SEM. ⴱ ⫽ P ⱕ 0.05 versus
CB1⫹/⫹ mice challenged with bleomycin.
significantly increased (by 29 ⫾ 6% and 18 ⫾ 3%,
respectively) in ACEA-treated mice (P ⫽ 0.05 and P ⫽
0.02, respectively) (Figures 2C and D). Thus, activation
of CB1 increases the sensitivity to bleomycin-induced
dermal fibrosis.
CB1 regulates leukocyte infiltration. Inflammatory infiltrates consisting mainly of T cells and macrophages are characteristic features of early stages of SSc
that are mimicked in the mouse model of bleomycininduced fibrosis. Infiltrating leukocytes stimulate fibroblast activation and collagen synthesis via the release of
profibrotic factors (1). Inflammatory infiltrates were
significantly reduced in CB1⫺/⫺ mice compared with
CB1⫹/⫹ mice challenged with bleomycin, and the number of infiltrating leukocytes was reduced by 63 ⫾ 7%
(P ⫽ 0.01) (Figure 3A). Further subanalyses revealed
that T cell and macrophage counts were significantly
lower in CB1⫺/⫺ mice, with decreases of 70 ⫾ 5% and
75 ⫾ 6%, respectively (P ⫽ 0.01 for each comparison)
(Figures 3B and C).
In contrast, treatment with the CB1 agonist
ACEA exacerbated the inflammatory response to bleomycin. The total number of infiltrating leukocytes as well
as T cell and macrophage counts increased significantly
upon treatment with ACEA compared with bleomycin
treatment alone (23 ⫾ 9% for the total number of
leukocytes [P ⫽ 0.05], 39 ⫾ 4% for T cells [P ⫽ 0.01],
and 50 ⫾ 3% for macrophages [P ⫽ 0.01]) (Figures
4A–C). Together, these data suggest that CB1 stimulates
leukocyte activation and tissue infiltration in experimental fibrosis.
CB1 on leukocytes is essential for the profibrotic
effects of CB1. The altered number of leukocytes in
lesional skin indicates that CB1 might regulate tissue
fibrosis by controlling leukocyte activation. To follow up
this hypothesis, bone marrow transplantation experiments were performed, and the response to bleomycin
was analyzed in the resulting chimeric mice. The number
of infiltrating leukocytes was not reduced in CB1⫺/⫺
mice reconstituted with CB1⫹/⫹ mouse bone marrow,
and these mice were not protected from bleomycininduced dermal fibrosis (further information is available
upon request from the corresponding author). Dermal
thickness, hydroxyproline content, and myofibroblast
counts in CB1⫺/⫺ mice with CB1⫹/⫹ mouse bone marrow were comparable with those observed in CB1⫹/⫹
mice with CB1⫹/⫹ mouse bone marrow (Figures 5A–D).
In contrast, CB1⫹/⫹ mice transplanted with bone
marrow cells from CB1⫺/⫺ mice were protected from
bleomycin-induced fibrosis. The numbers of T cells and
macrophages as well as the total number of leukocytes in
lesional skin of CB1⫹/⫹ mice with CB1⫺/⫺ mouse bone
marrow were reduced to a degree similar to that observed in CB1⫺/⫺ mice with CB1⫺/⫺ mouse bone mar-
3472
MARQUART ET AL
Figure 4. Leukocyte infiltration into lesional skin is regulated by CB1. Shown are increased leukocyte infiltration (A) with higher
numbers of T cells (B) and macrophages (C) in mice treated with the CB1 agonist N-(2-chloroethyl)-5Z,8Z,11Z,14Zeicosatetraenamide (ACEA). CB1⫹/⫹ control mice were injected with NaCl, and the values for these mice were set at 1.0; the other
results were normalized to this value. Values are the mean ⫾ SEM. ⴱ ⫽ P ⱕ 0.05 versus CB1⫹/⫹ mice challenged with bleomycin alone.
row (further information is available upon request from
the corresponding author). Moreover, dermal thickening in CB1⫹/⫹ mice with CB1⫺/⫺ mouse bone marrow
did not differ from that observed in CB1⫺/⫺ mice with
CB1⫺/⫺ mouse bone marrow (increases of 46 ⫾ 4% and
39 ⫾ 4%, respectively; P ⫽ 0.34), but was significantly
lower than that in CB1⫹/⫹ mice with CB1⫹/⫹ mouse
bone marrow (increase of 103 ⫾ 3%) (P ⫽ 0.01)
Figure 5. Inactivation of CB1 on leukocytes completely mimics the antifibrotic effects observed in CB1-deficient (CB1⫺/⫺) mice. A,
Bone marrow (BM) transplantation experiments revealed that CB1⫹/⫹ mice transplanted with CB1⫺/⫺ mouse bone marrow were
protected from bleomycin-induced dermal fibrosis to a degree similar to that of CB1⫺/⫺ mice with CB1⫺/⫺ mouse bone marrow,
whereas no antifibrotic effects were observed in CB1⫺/⫺ mice with CB1⫹/⫹ mouse bone marrow. Representative skin sections are
shown (hematoxylin and eosin stained; original magnification ⫻ 100). B–D, Shown are comparable reductions of dermal thickening
(B), hydroxyproline content (C), and myofibroblast counts (D) between CB1⫹/⫹ mice transplanted with bone marrow from CB1⫺/⫺
mice and CB1⫺/⫺ mice with CB1⫺/⫺ mouse bone marrow upon challenge with bleomycin. The values for CB1⫹/⫹ mice with CB1⫹/⫹
mouse bone marrow are set at 100%; other results are shown as changes relative to these mice. Values are the mean ⫾ SEM. ⴱ ⫽ P ⱕ
0.05 versus bleomycin-challenged CB1⫹/⫹ mice with CB1⫹/⫹ mouse bone marrow.
ANTIFIBROTIC EFFECTS OF CB1 INACTIVATION
3473
Figure 6. Inactivation of CB1 does not reduce fibrosis in TSK-1 mice. TSK-1 mice are a model of inflammation-independent fibrosis without
inflammatory infiltrates and with endogenous activation of fibroblasts. Consistent with the hypothesis that inactivation of CB1 exerts its
antifibrotic effects by inhibiting leukocyte infiltration, but not by direct effects on fibroblasts, CB1-deficient (CB1⫺/⫺) TSK-1 mice were not
protected from fibrosis (original magnification ⫻ 100) (A). Hypodermal thickness (B), hydroxyproline content (C), and myofibroblast counts (D)
did not differ between CB1⫺/⫺ TSK-1 mice and their CB1⫹/⫹ TSK-1 littermates. Values for CB1⫹/⫹ pa/pa (control) mice were set at 1.0; all other
results were normalized to this value. Values are the mean ⫾ SEM.
(Figures 5A and B). Consistently, hydroxyproline content and myofibroblast counts in CB1⫹/⫹ mice reconstituted with CB1⫺/⫺ mouse bone marrow were similar to
those in CB1⫺/⫺ mice with CB1⫺/⫺ mouse bone marrow,
but were significantly reduced compared with those in
CB1⫹/⫹ mice with CB1⫹/⫹ mouse bone marrow (Figures
5C and D). These data demonstrate that CB1 exerts its
profibrotic effects indirectly by regulating leukocyte
infiltration.
Inhibition of CB1 does not prevent inflammationindependent fibrosis. To confirm that CB1 controls
fibroblast activation indirectly by regulating leukocyte
infiltration, we analyzed the role of CB1 in the TSK-1
mouse model of SSc. Inflammatory infiltrates are absent
in TSK-1 mice, and the increased release of collagen by
TSK-1 mouse fibroblasts is caused by endogenous activation and not by the release of profibrotic cytokines
from infiltrating leukocytes as in bleomycin-induced
fibrosis (20,25). Consistent with the results from bone
marrow transplantation and with the hypothesis that
inactivation of CB1 exerts its antifibrotic effects
by inhibiting leukocyte infiltration and not by direct
inhibitory effects on fibroblasts, CB1⫺/⫺ TSK-1 mice
were not protected from fibrosis. No significant differences in hypodermal thickness, hydroxyproline content,
and myofibroblast counts were observed between
CB1⫺/⫺ TSK-1 mice and their CB1⫹/⫹ TSK-1 littermates (Figures 6A–D).
DISCUSSION
Fibrosis is a major reason for the high morbidity
of SSc patients and is also a leading cause of their
increased mortality (26). Treatment of fibrosis is a major
challenge for physicians, because efficient antifibrotic
therapies that target specifically the aberrant activation
of SSc fibroblasts are currently not available for clinical
use (27). Inactivation of CB1 has previously been shown
in an elegant study to decrease the wound healing
response to acute liver injury and to inhibit progression
of liver cirrhosis (28). Our data constitute the first
evidence that the effects of CB1 are not restricted to the
liver but are also operative in other fibrotic diseases with
significant differences in the histologic changes and in
the underlying pathogenesis.
The pathogenesis of skin fibrosis differs from that
of liver cirrhosis in several ways, including but not
limited to 1) the lack of stellate cells in the skin that are
thought to play a key role in the pathogenesis of liver
cirrhosis; 2) no role of epidermal-to-mesenchymal transition in the skin, in contrast to the prominent role of
this transition in liver cirrhosis; and 3) the unclear role of
3474
bone marrow–derived fibrocytes in the pathogenesis of
SSc. These differences are also reflected in differences
in genes up-regulated during fibrogenesis and in their
expression pattern. These differences in pathogenesis
are also reflected by different mechanisms of action of
CB1 in SSc and liver cirrhosis. In liver cirrhosis, CB1
seems to exert its profibrotic effects by inhibiting proliferation of hepatic myofibroblasts. In contrast, in experimental SSc we demonstrate that the profibrotic effects
are exclusively mediated by the effects of CB1 on
leukocytes, and we demonstrate that inhibition of CB1
prevents leukocyte infiltration into lesional skin.
Although additional studies on more complex
murine models of SSc, such as in Fra-2–transgenic mice
or in caveolin 1–knockout mice (23,29,30), are needed
for final conclusions, our findings might have clinical
implications, since specific inhibitors of CB1, such as
SR141716A, have been developed and have been proven
to potently inhibit CB1 signaling in humans (13,14).
However, the incidence of depression and fear was
increased in patients receiving SR141716A, and suicidal
behavior might occur in predisposed patients. It is
currently unknown whether these adverse effects were a
class effect or were specific for SR141716A. However,
these reports warrant a careful monitoring of patients
receiving CB1 antagonists.
The 2 cannabinoid receptors, CB1 and CB2, have
opposing roles in fibrosis. Inhibition of CB2 exacerbates
fibrosis, whereas agonists of CB2 exert antifibrotic effects in different models (15,31,32). In contrast, we
demonstrate in the present study that CB1 is profibrotic
and that activation of CB1 exerts profibrotic effects,
whereas inhibition of CB1 prevents bleomycin-induced
dermal fibrosis. These results warrant the use of highly
selective drugs that either inhibit CB1 or activate CB2
but that do not affect the other cannabinoid receptor.
Nonspecific drugs such as the CB1/CB2 agonist dronabinol, which is approved for the treatment of cachexia and
of nausea resulting from chemotherapy, act on both
cannabinoid receptors, and their effects on fibrosis are
unpredictable. The antifibrotic effects caused by the
activation of CB2 might be counterbalanced or even
outweighed by the profibrotic effects of the activation of
CB1. It will also be interesting to determine the role of
endocannabinoids in fibrosis. These endogenous agonists often bind CB1 and CB2, but with different affinities (33). Will the profibrotic effects of the activation of
CB1 dominate, or will endocannabinoids preferentially
activate CB2 receptors and exert antifibrotic effects?
Early stages of SSc are characterized by an
infiltration of affected skin by inflammatory cells, in
MARQUART ET AL
particular T cells and macrophages (1,2). The infiltrating
leukocytes release profibrotic cytokines, such as monocyte chemoattractant protein 1, interleukin-4 (IL-4), and
IL-13, that stimulate the collagen synthesis in resident
fibroblasts (2,34,35). CB1 might play a crucial role in
these early, inflammatory stages of SSc. Our results
demonstrate that CB1 stimulates the infiltration of
leukocytes into lesional skin in inflammation-driven
models of fibrosis. Knockdown of CB1 significantly
reduced the numbers of leukocytes in bleomycinchallenged mice, whereas activation of CB1 resulted in
increased leukocyte infiltration. These findings indicate
that inhibition of CB1 exerts its antifibrotic effects
indirectly by orchestrating the infiltration of leukocytes
into lesional skin rather than by direct effects on the
collagen synthesis of fibroblasts. Consistent with this
hypothesis, the release of collagen was not altered in
fibroblasts isolated from CB1⫺/⫺ mice (data not shown).
Moreover, the antifibrotic effects caused by general
inactivation of CB1 fully resembled those caused by
transplantation of CB1⫺/⫺ mouse bone marrow cells
into WT mice. In contrast, no protective effect was
observed in CB1⫺/⫺ mice reconstituted with bone marrow cells from WT mice. Finally, inactivation of CB1 did
not exert antifibrotic effects in the TSK-1 mouse model,
which serves as an inflammation-independent model
with endogenous fibroblast activation.
WIN55,212-2, a nonselective, synthetic cannabinoid receptor agonist, was recently reported to decrease
the release of collagen in cultured fibroblasts (36).
However, the concentrations of WIN55,212-2 used in
that study were very high and induced apoptosis in
fibroblasts. Thus, the reduced synthesis of collagen upon
incubation with WIN55,212-2 might have been due to
toxic effects rather than to specific antifibrotic effects.
Together, these data suggest that CB1 positively regulates the infiltration of leukocytes and enhances their
migration into lesional skin in SSc. Inactivation of CB1
reduces leukocyte infiltration and prevents the release of
profibrotic mediators, which results in decreased activation of resident fibroblasts and protection from fibrosis.
CB1 might regulate leukocyte activation on different
levels. Besides the effects on migration of leukocytes,
blockade of CB1 might shift the balance from a Th2-type
to a Th1-type immune response. Activation of CB1
increased the release of the Th2 cytokine IL-4 and
suppressed the levels of the Th1 cytokines interferon-␥
and IL-12 (37). Pharmacologic inhibition of CB1 shifted
the immune response from a Th2-type to a Th1-type
response. Since Th2 cytokines such as IL-4 potently
stimulate fibroblasts to release extracellular matrix pro-
ANTIFIBROTIC EFFECTS OF CB1 INACTIVATION
teins (38,39), prevention of Th2 differentiation might
play a major role in the antifibrotic effects observed
upon inactivation of CB1.
In summary, we demonstrate that CB1 indirectly
regulates the activation of fibroblasts by orchestrating
the influx of leukocytes into lesional skin. Activation of
CB1 enhanced leukocyte infiltration and inflammationdriven fibrosis, whereas ablation of CB1 exerted potent
antifibrotic effects. Thus, CB1 might be a potential molecular target for the treatment of inflammatory stages
of SSc.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. J. H. W. Distler had full access to
all of the data in the study and takes responsibility for the integrity of
the data and the accuracy of the data analysis.
Study conception and design. Marquart, Zwerina, O. Distler, Schett,
J. H. W. Distler.
Acquisition of data. Marquart, Zerr, Palumbo, Reich, Tomcik, Horn,
Dees, Engel, J. H. W. Distler.
Analysis and interpretation of data. Marquart, Akhmetshina,
J. H. W. Distler.
REFERENCES
1. Gabrielli A, Avvedimento EV, Krieg T. Scleroderma. N Engl
J Med 2009;360:1989–2003.
2. Varga J, Abraham D. Systemic sclerosis: a prototypic multisystem
fibrotic disorder. J Clin Invest 2007;117:557–67.
3. Kahari VM, Sandberg M, Kalimo H, Vuorio T, Vuorio E. Identification of fibroblasts responsible for increased collagen production in localized scleroderma by in situ hybridization. J Invest
Dermatol 1988;90:664–70.
4. Mackie K. Cannabinoid receptors: where they are and what they
do. J Neuroendocrinol 2008;20 Suppl 1:10–4.
5. Bisogno T. Endogenous cannabinoids: structure and metabolism.
J Neuroendocrinol 2008;20 Suppl 1:1–9.
6. Sarfaraz S, Adhami VM, Syed DN, Afaq F, Mukhtar H. Cannabinoids for cancer treatment: progress and promise. Cancer Res
2008;68:339–42.
7. Bab I, Zimmer A. Cannabinoid receptors and the regulation of
bone mass. Br J Pharmacol 2008;153:182–8.
8. Klein TW. Cannabinoid-based drugs as anti-inflammatory therapeutics. Nat Rev Immunol 2005;5:400–11.
9. Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bonner TI.
Structure of a cannabinoid receptor and functional expression of
the cloned cDNA. Nature 1990;346:561–4.
10. Munro S, Thomas KL, Abu-Shaar M. Molecular characterization
of a peripheral receptor for cannabinoids. Nature 1993;365:61–5.
11. Idris AI, van ’t Hof RJ, Greig IR, Ridge SA, Baker D, Ross RA,
et al. Regulation of bone mass, bone loss and osteoclast activity by
cannabinoid receptors. Nat Med 2005;11:774–9.
12. Ofek O, Karsak M, Leclerc N, Fogel M, Frenkel B, Wright K, et al.
Peripheral cannabinoid receptor, CB2, regulates bone mass. Proc
Natl Acad Sci U S A 2006;103:696–701.
13. Bifulco M, Santoro A, Laezza C, Malfitano AM. Cannabinoid
receptor CB1 antagonists state of the art and challenges. Vitam
Horm 2009;81:159–89.
14. Scheen AJ, Paquot N. Use of cannabinoid CB1 receptor antago-
3475
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
nists for the treatment of metabolic disorders. Best Pract Res Clin
Endocrinol Metab 2009;23:103–16.
Akhmetshina A, Dees C, Busch N, Beer J, Sarter K, Zwerina J,
et al. The cannabinoid receptor CB2 exerts antifibrotic effects in
experimental dermal fibrosis. Arthritis Rheum 2009;60:1129–36.
Marsicano G, Wotjak CT, Azad SC, Bisogno T, Rammes G,
Cascio MG, et al. The endogenous cannabinoid system controls
extinction of aversive memories. Nature 2002;418:530–4.
Distler JH, Jungel A, Huber LC, Schulze-Horsel U, Zwerina J,
Gay RE, et al. Imatinib mesylate reduces production of extracellular matrix and prevents development of experimental dermal
fibrosis. Arthritis Rheum 2007;56:311–22.
Hillard CJ, Manna S, Greenberg MJ, DiCamelli R, Ross RA,
Stevenson LA, et al. Synthesis and characterization of potent and
selective agonists of the neuronal cannabinoid receptor (CB1).
J Pharmacol Exp Ther 1999;289:1427–33.
Luszczki JJ, Czuczwar P, Cioczek-Czuczwar A, Czuczwar SJ.
Arachidonyl-2’-chloroethylamide, a highly selective cannabinoid
CB1 receptor agonist, enhances the anticonvulsant action of valproate in the mouse maximal electroshock-induced seizure model.
Eur J Pharmacol 2006;547:65–74.
Green MC, Sweet HO, Bunker LE. Tight-skin, a new mutation of
the mouse causing excessive growth of connective tissue and
skeleton. Am J Pathol 1976;82:493–512.
Akhmetshina A, Dees C, Pileckyte M, Maurer B, Axmann R,
Jungel A, et al. Dual inhibition of c-abl and PDGF receptor
signaling by dasatinib and nilotinib for the treatment of dermal
fibrosis. FASEB J 2008;22:2214–22.
Woessner JF Jr. The determination of hydroxyproline in tissue and
protein samples containing small proportions of this imino acid.
Arch Biochem Biophys 1961;93:440–7.
Reich N, Maurer B, Akhmetshina A, Venalis P, Dees C, Zerr P,
et al. The transcription factor Fra-2 regulates the production of
extracellular matrix in systemic sclerosis. Arthritis Rheum 2010;
62:280–90.
Skhirtladze C, Distler O, Dees C, Akhmetshina A, Busch N, Venalis
P, et al. Src kinases in systemic sclerosis: central roles in fibroblast
activation and in skin fibrosis. Arthritis Rheum 2008;58:1475–84.
Akhmetshina A, Venalis P, Dees C, Busch N, Zwerina J, Schett G,
et al. Treatment with imatinib prevents fibrosis in different
preclinical models of systemic sclerosis and induces regression of
established fibrosis. Arthritis Rheum 2009;60:219–24.
Chung L, Krishnan E, Chakravarty EF. Hospitalizations and
mortality in systemic sclerosis: results from the Nationwide Inpatient Sample. Rheumatology (Oxford) 2007;46:1808–13.
Charles C, Clements P, Furst DE. Systemic sclerosis: hypothesisdriven treatment strategies. Lancet 2006;367:1683–91.
Teixeira-Clerc F, Julien B, Grenard P, Tran Van Nhieu J, Deveaux
V, Li L, et al. CB1 cannabinoid receptor antagonism: a new
strategy for the treatment of liver fibrosis. Nat Med 2006;12:671–6.
Del Galdo F, Sotgia F, de Almeida CJ, Jasmin JF, Musick M,
Lisanti MP, et al. Decreased expression of caveolin 1 in patients
with systemic sclerosis: crucial role in the pathogenesis of tissue
fibrosis. Arthritis Rheum 2008;58:2854–65.
Maurer B, Busch N, Jungel A, Pileckyte M, Gay RE, Michel BA,
et al. Transcription factor fos-related antigen-2 induces progressive peripheral vasculopathy in mice closely resembling human
systemic sclerosis. Circulation 2009;120:2367–76.
Julien B, Grenard P, Teixeira-Clerc F, Van Nhieu JT, Li L, Karsak
M, et al. Antifibrogenic role of the cannabinoid receptor CB2 in
the liver. Gastroenterology 2005;128:742–55.
Michalski CW, Maier M, Erkan M, Sauliunaite D, Bergmann F,
Pacher P, et al. Cannabinoids reduce markers of inflammation and
fibrosis in pancreatic stellate cells. PLoS ONE 2008;3:e1701.
Klein TW, Newton CA. Therapeutic potential of cannabinoidbased drugs. Adv Exp Med Biol 2007;601:395–413.
Distler JH, Akhmetshina A, Schett G, Distler O. Monocyte
3476
chemoattractant proteins in the pathogenesis of systemic sclerosis.
Rheumatology (Oxford) 2009;48:98–103 [review].
35. Distler O, Pap T, Kowal-Bielecka O, Meyringer R, Guiducci S,
Landthaler M, et al. Overexpression of monocyte chemoattractant
protein 1 in systemic sclerosis: role of platelet-derived growth
factor and effects on monocyte chemotaxis and collagen synthesis.
Arthritis Rheum 2001;44:2665–78.
36. Garcia-Gonzalez E, Selvi E, Balistreri E, Lorenzini S, Maggio R,
Natale MR, et al. Cannabinoids inhibit fibrogenesis in diffuse systemic sclerosis fibroblasts. Rheumatology (Oxford) 2009;48:1050–6.
MARQUART ET AL
37. Klein TW, Newton C, Larsen K, Chou J, Perkins I, Lu L, et al.
Cannabinoid receptors and T helper cells. J Neuroimmunol 2004;147:
91–4.
38. Distler JH, Jungel A, Caretto D, Schulze-Horsel U, KowalBielecka O, Gay RE, et al. Monocyte chemoattractant protein 1
released from glycosaminoglycans mediates its profibrotic effects
in systemic sclerosis via the release of interleukin-4 from T cells.
Arthritis Rheum 2006;54:214–25.
39. Wynn TA. Fibrotic disease and the T(H)1/T(H)2 paradigm. Nat
Rev Immunol 2004;4:583–94.
Документ
Категория
Без категории
Просмотров
0
Размер файла
337 Кб
Теги
prevent, experimentov, cannabinoid, inactivation, leukocytes, infiltrating, fibrosis, receptov, cb1
1/--страниц
Пожаловаться на содержимое документа